Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular amastigote forms within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandflies), they differentiate and multiply as, extracellular promastigote forms within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani). Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Such secretory products can readily permeate throughout and presumably alter the host micro-environments in which Leishmania reside. Thus, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes and regulatory proteins are being investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized toward defining their expression and regulation during parasite growth, development and differentiation. For example, recently we identified and characterized the genes that encode the unique Leishmania secretory chitinase family. Using combined biochemical and molecular approaches we showed that this family of enzymes was functionally conserved among all pathogenic species of Leishmania examined. This suggested that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In that regard, in on going collaborative studies, we have also isolated, characterized and expressed the gene encoding the L. mexicana chitinase. Further, we have examined the phenotype of L. mexicana chitinase over-expressor transfectants in their sandfly vectors. Our observations indicate that these organisms are able to egress from the host peritrophic membrane compartment and infect the sandfly host at significantly elevated levels vs controls. In addition, such transfectants cause greater damage to the fly stomadeal valve system which results in greater numbers of parasites being transmitted to their host. These results indicate that this secretory enzyme plays essential roles in the development and transmission of this parasite it its sanfly vector. In other on going studies, attempts are being made to functionally delete the gene for the L. mexicana chitinase and to examine the viability of these mutants in both the mammalian (mouse) host and sandfly vectors. In other studies, we are evaluating whether vaccination of mice with cDNA of the Leishmanial chitinase will result in alteration of parasite pathology following challenge infections with virulent parasites. In addition, previously, we demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. In that regard, in parallel ongoing studies, we recently identified and characterized the biochemical and functional properties of a unique new, 35 kDa, nuclease from Leishmania donovani. Our studies demonstrated that this enzyme was constitutively released/secreted by both amastigotes and promastigote developmental forms of this parasite. Using a molecular approach, we identified, characterized and episomally-expressed the gene, LdNucS which encodes this new Class I nuclease family member from these organisms. Biochemical and enzymatic characterization showed that the LdNucS nuclease was capable of cleaving a variety of synthetic polynucleotide substrates as well as effectively hydrolyzing natural RNA, single stranded and double stranded DNA substrates. We hypothesize that this leishmanial secretory nuclease could act, at a distance away from the parasite, to hydrolyze host-derived nucleic acids to satisfy the essential purine requirements of these organisms. This suggests that it must have essential role(s) in facilitating the survival, growth and development of this important human pathogen in both its insect vector and mammalian hosts. In support of the latter, we have recently demonstrated that patients from multiple diverse geographic endemic foci all appear to posses antibodies which react with and immunoprecipitate the Leishmania LdNucS nuclease. These observations suggest that this enzyme is synthesized and released by the parasite during the course of human infections. In that regard, this leishmanial secretory nuclease might be exploited for diagnostic or for therapeutic purposes.[unreadable] Taken together, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.